Cytokine receptor genes and the use thereof to enhance therapy

ABSTRACT

Cells, such a T-cells, are provided that comprise cytokine receptors having increased activity in response to their ligand. For example, cell can comprise IL-2 and/or IL-15 receptors having increased surface expression or signaling activity. Cells of the embodiments have a significant growth advantage in the presence of cytokines and can be used, e.g., for enhanced adoptive cell transfer therapies.

This application claims the benefit of U.S. Provisional PatentApplication No. 62/106,860, filed Jan. 23, 2015 and 62/242,098, filedOct. 15, 2015, both of which are incorporated herein by reference.

GOVERNMENT SUPPORT CLAUSE

The invention was made with government support under Grant No.P01CA54778-01 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates generally to the fields of molecularbiology, immunology and medicine. In particular, embodiments of theinvention relate to transferring of cytokine receptor genes ormodulation of cytokine receptor gene expression to enhance sensitivityto cytokine therapy.

2. Description of Related Art

Recently, there has been increasing interest in cell-based therapies totreat disease. For example, therapies using T-cells targeted to tumorantigens, such as chimeric antigen receptor T-cells, are being studiedas potential new anti-cancer therapies. However, for such therapies tobe effective, the therapeutic cells must be able to proliferate in vivo.Accordingly, many of these therapies rely upon administration ofexogenous cytokines, such as IL-2, to the patients being treated. Theadministration of IL-2 and many other cytokines/protein therapeutics isoften limited by dose limiting toxicity. In the case of IL-2, forexample, therapeutic effector cells may respond inefficiently to thecytokine. Thus, there remains a need for therapeutic cells with enhancedproliferative capability or enhanced response to cytokines.

SUMMARY OF THE INVENTION

In a first embodiment there is provided an isolated cell comprising (i)an elevated surface expression level of at least one cytokine receptoror cytokine receptor co-stimulator (e.g., relative to an activated ornaïve T-cell); or (ii) a nucleic acid molecule encoding at least onecytokine receptor or cytokine receptor co-stimulator polypeptidecomprising a mutation that increases the activity of the receptor. Forexample, the at least one cytokine receptor can be selected from IL-2R(a receptor for IL-2), IL-15R (a receptor for IL-15), IL-12R (a receptorfor IL-12), IL-6R (a receptor for IL-6), GP130 (a receptor for IL-6,IL-35 or IL-27), IL-4R (a receptor for IL-4), IL-2Rγ (a receptor forIL-4 or IL-9), IL-27R (a receptor for IL-27), IL-2Rβ2 (a receptor forIL-35), IL-12Rβ1 (a receptor for IL-23), IL-23R (a receptor for IL-23),IL-9R (a receptor for IL-9), G-CSFR (a receptor for G-CSF and theneupogen or neulasta ligands) or GM-CSF (a receptor for GM-CSF). In afurther aspects, the cytokine receptor co-stimulator is ICOS (whichbinds to ICOS ligand), 4-1BB (which binds to 4-1BB ligand) or CD28(which binds to B7-1 or B7-2). In some aspects, the cell is a mammaliancell, such as a human cell. In further aspects, the cell is an immunecell, such as a T-cell, Natural Killer (NK) cell or NK T-cell. Inspecific aspects, the T-cell is a cytotoxic T-cell, an inflammatoryT-cell, an effector T-cell or a memory T-cell. In further aspects, theT-cell is a CD4⁺ or CD8⁺ T-cell. In further aspects, the T cell is analpha/beta T cell, a gamma/delta T cell, NK T cell, or other lymphocytesubpopulation. In still further aspects, the cell is comprised in a bonemarrow graft cell population.

Thus, in a further embodiment there is provided an isolated transgeniccell comprising (i) an elevated surface expression level of a cytokinereceptor; or (ii) a nucleic acid molecule encoding a cytokine receptorpolypeptide comprising a mutation that increases the activity of thereceptor (e.g., when bound to the cytokine ligand). For example, in someaspects, the cytokine receptor is proliferative cytokine receptor, suchas an IL-2 receptor (e.g., IL-2Rα), GM-CSF receptor (e.g., GM-CSFRα orGM-CSFRβc), G-CSF receptor, IL-12 receptor (e.g., IL-12Rβ1 or IL-12Rβ2)or an IL-15 receptor (e.g., IL-15Rα).

In further embodiment there is provided an isolated transgenic cell,such as a T-cell, comprising (i) an elevated surface expression level ofan IL-2 receptor; or (ii) a nucleic acid molecule encoding an IL-2receptor polypeptide comprising a mutation that increases the activityof the receptor. For example, the elevated surface expression level ofan IL-2 receptor, can be elevated relative to the expression levelexhibited by an activated T-cell. In further aspects of the invention,expression will be elevated versus a naïve or unactivated T cells. Instill further aspects, a cell of the embodiments may comprise IL-2Rexpression that is maintained in culture conditions that would normallydown regulate IL-2R. In certain aspects, an IL-2 receptor polypeptide ofthe embodiments is an IL-2Rα, IL-2Rβ and/or IL-2Rγ polypeptide.

Thus, in specific aspects, an isolated cell of the embodiments comprisesa nucleic acid molecule encoding an IL-2 receptor polypeptide comprisinga mutation that increases the activity of the receptor. In particularaspects, the IL-2 receptor is IL-2Rα (also referred to as CD25). Inother aspects, the IL-2 receptor is IL-2Rβ (also referred to as CD122)or IL-2Rγ (also referred to as CD132). In some aspects, the mutationincrease surface expression, increases stability or increases ligandbinding of the IL-2 receptor polypeptide. In certain aspects, themutation disrupts one or more ribosylation sites on IL2-Rα (see, e.g.,Teege et al., 2015, incorporated herein by reference). In furtheraspects, the signal sequence for recycling and/or endosomal sorting ofthe IL-2 receptor is modified to alter the natural distribution orre-expression of the receptor (see, e.g., Amano et al., 2013,incorporated herein by reference). In still further aspects, a nucleicacid molecule encoding the IL-2 receptor polypeptide is operably linkedto a heterologous promoter. In certain aspects, the heterologouspromoter is a ligand inducible or a ligand repressible promoter. In someaspects, the ligand inducible promoter is a tet-on promoter. In certainaspects, the nucleic acid molecule encoding the IL-2 receptorpolypeptide is integrated into the genome of the cell or is encoded onan episomal vector. In further specific aspects, the nucleic acidmolecule encoding the IL-2 receptor polypeptide is flanked by retrovirallong terminal repeats or transposon repeats.

In a further embodiment there is provided an isolated transgenic cell,such as a T-cell, comprising (i) an elevated surface expression level ofan IL-15 receptor; or (ii) a nucleic acid molecule encoding an IL-15receptor polypeptide comprising a mutation that increases the activityof the receptor. For example, the elevated surface expression level ofan IL-15 receptor, can be elevated relative to the expression levelexhibited by an activated T-cell. In further aspects of the invention,expression will be elevated versus a naïve or unactivated T cells. Instill further aspects, a cell of the embodiments may comprise IL-15Rexpression that is maintained in culture conditions that would normallydown regulate IL-15R. In certain aspects, an IL-15 receptor polypeptideof the embodiments is an IL-15Rα, IL-2Rβ and/or IL-2Rγ polypeptide.

Thus, in specific aspects, an isolated cell of the embodiments comprisesa nucleic acid molecule encoding an IL-15 receptor polypeptidecomprising a mutation that increases the activity of the receptor. Inparticular aspects, the IL-15 receptor is IL-15Rα. In other aspects, theIL-15 receptor is IL-2Rβ or IL-2Rγ. In some aspects, the mutationincrease surface expression, increases stability or increases ligandbinding of the IL-15 receptor polypeptide. In further aspects, thesignal sequence for recycling and/or endosomal sorting of the IL-15receptor is modified to alter the natural distribution or re-expressionof the receptor (see, e.g., Amano et al., 2013, incorporated herein byreference). In still further aspects, a nucleic acid molecule encodingthe IL-15 receptor polypeptide is operably linked to a heterologouspromoter. In certain aspects, the heterologous promoter is a ligandinducible or a ligand repressible promoter. In some aspects, the ligandinducible promoter is a tet-on promoter. In certain aspects, the nucleicacid molecule encoding the IL-15 receptor polypeptide is integrated intothe genome of the cell or is encoded on an episomal vector. In furtherspecific aspects, the nucleic acid molecule encoding the IL-15 receptorpolypeptide is flanked by retroviral long terminal repeats or transposonrepeats.

In yet still a further aspect of the above embodiments, an isolated cellcomprises a further transgene, such as a suicide gene, a chimericantigen receptor (CAR) or a recombinant T-cell receptor (TCR). Such afurther transgene may be encoded on the same nucleic acid molecule as acytokine receptor of the embodiments or may be encoded on a separatemolecule. In some aspects, the further transgene is a suicide gene thatis operably linked to an inducible promoter. For example, the suicidegene can be a thymidine kinase gene. In further aspects a cell of theembodiments comprises a cell surface marker. For example, the cell cancomprise a marker such as CD20 that can be depleted by antibodyadministration.

In some aspects, an isolated cell of the embodiments is a T-cell or anNK-cell targeted to an infectious disease or cancer cell antigen. Forexample, the cancer cell antigen can be an oncogene or a growth factorreceptor. In particular aspects, the cancer cell antigen is CD19, CD20,GP240, 5T4, HER1, CD-33, CD-38, VEGFR-1, VEGFR-2, CEA, FGFR3, IGFBP2,IGF-1R, BAFF-R, TACI, APRIL, Fn14, EGFR, ERBB2, ERBB3 or mesothelin. Infurther aspects, the isolated cell expresses a chimeric antigen receptor(CAR) or a recombinant T-cell receptor (TCR) targeted to an infectiousdisease or cancer cell antigen.

In a further embodiment there is provided a pharmaceutical compositioncomprising an isolated cell in accordance with any of the embodimentsand aspects described above in a pharmaceutically acceptable carrier. Insome aspects, the composition comprises between about 1×10³ and 1×10⁴,1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹ or 1×10¹² cells inaccordance with any of the embodiments and aspects described above.

In still a further embodiment, there is provided a method of providing aT-cell response in a human subject having a disease comprisingadministering an effective amount of T-cells having increased expressionor activity of a cytokine receptor, as described above, to the subject.In certain aspects, the T-cell response is a regulatory T-cell response.In other aspects, the T-cell response is a cytotoxic T-cell response. Infurther aspects, the method further comprises administering a cytokinethat stimulates T-cell proliferation to the subject. In particularaspects, the cytokine is IL-2 or IL-15. In some aspects, the cytokineadministered to the subject (e.g., IL-2 or IL-15) comprises a mutationthat increases receptor binding. In some aspects, the cytokine has beenmodified to increase serum half-life. For example, the cytokine can bePEGylated or fused to an Fc polypeptide. In a further aspect, thecytokine may be bound to an antibody or soluble receptor. For example,IL-2 may be bound to anti-IL-2 mAb or IL-15 may be bound to solubleIL-15Rα with or without an Fc fusion (see, e.g., Boyman et al., 2006 andRubinstein et al., 2006, each of which are incorporated herein byreference).

In yet a further embodiment there is provided a method for controlling aT-cell response in a subject. In some aspects, such a method comprisesidentifying a subject who has been treated with a cell population of theembodiments (e.g., cells comprising increased cytokine receptoractivity, such as increased IL-2R activity) and administering atherapeutic the subject that selectively kills or inhibits proliferationof the cell population. For example, in the case of a cell populationhaving increased IL-2R activity, a subject can be administered anIL-2-like molecule that is fused or conjugated to a toxin (such as ricinor gelonin). In some aspects, the IL-2-like molecule preferentiallybinds cells with elevated IL-2Rα expression and leads to theirdestruction or depletion. In further aspects, a subject can beadministered an antibody to the cytokine receptor having increasedactivity. For example, a subject can be administered an antibody againstIL-2Rα (or other receptor subunit protein) to deplete or destroy cellsthat are no longer desirable. For instance, the antibody could bedaclizumab (anti-IL-2Rα mAb).

In yet still a further embodiment, there is provided a method ofproducing therapeutic cells comprising: (i) selecting a population ofcells having increased cytokine receptor activity (or cytokineco-receptor activity), for a proliferative cytokine receptor; and (ii)culturing the cells in the presence of a ligand for the proliferativecytokine receptor. In further aspects, the method may further comprise(i) selecting a population of cells having increased cytokine receptoractivity (or cytokine co-receptor activity), for a proliferativecytokine receptor; (ii) culturing the cells in the presence of a ligandfor the proliferative cytokine receptor, thereby producing an expandedcall population; and (iii) selecting cells from the expanded populationthat do not have increased cytokine receptor activity. For example, anexpanded cell population can be treated with and agent that reduces theactivity of expression level of the cytokine receptor. In a furtheraspect, the method comprises (i) selecting a population of cells havingincreased IL-2 or IL-15 receptor activity; and (ii) culturing the cellsin the presence of IL-2 or IL-15. In certain aspects, the cells havingincreased IL-2 or IL-15 receptor activity express or an elevated levelof surface IL-2 or IL-15 receptor on their surface. In further aspects,selecting a population of cells having increased IL-2 or IL-15 receptoractivity comprises contacting the cells a drug that increases IL-2 orIL-15 receptor expression. In certain aspects, selecting a population ofcells having increased IL-2 or IL-15 receptor activity comprises sortingcells based on IL-2 or IL-15 receptor expression. In some particularaspects, the cells comprise a nucleic acid molecule encoding an IL-2 orIL-15 receptor polypeptide comprising a mutation that increases theactivity of the receptor.

In a further embodiment, there is provided a method of producing cellswith elevated receptor or receptor subunit expression (e.g., usingmethods not requiring the transfer of genetic material). For example,therapeutic cells can be cultured with pharmacological agents beforeadoptive transfer and/or the pharmacological agents can be administeredduring or after adoptive transfer. For example, the pharmacologicalagents can include, without limitation, cytokines, agonists forco-stimulatory molecules, epigenetic drugs, or related compounds. Thus,therapeutic cells can be cultured in a cytokine such as IL-12 and/orIL-18 before adoptive transfer. For example, the therapeutic cells canbe cultured in a cytokine such as IL-21 before adoptive transfer. Infurther aspects, the therapeutic cells are cultured with an agonistagainst co-stimulatory molecules such as CD28, ICOS, or 4-1BB. In yetfurther aspects, the therapeutic cells are cultured with epigeneticdrugs that target certain pathways, such as HDAC2 and G9a which repressexpression of IL-2Ra (see, e.g., Shin et al., 2013, which isincorporated herein by reference).

In yet a further embodiment, there is provided a method of producingcells with elevated receptor or receptor subunit expression (e.g., usingmethods not requiring the transfer of genetic material) comprisingsorting or enriching cells for a receptor or a receptor subunitexpression prior to culture, during culture, or immediately prior toadoptive transfer. For example, cell can be sorted or selected using anantibody against IL-2Rα (e.g., by fluorescence assisted cell sorting(FACS), column purification or bead sorting of cells expressing elevatedlevels of IL-2Rα). Such sorted cells may be cultured for an additionalperiod of time or immediately adoptively transferred. This method canoptionally be used with cell and methods of the embodiments describedherein above. In some aspects, this methodology has the added benefit ofallowing the receptor to be used as selectable marker to enrich orisolate genetically modified T cells.

In still a further embodiment there is provided a method of treating adisease comprising transferring at least one receptor gene into at leastone cell and treating said cell or cells with an agonist of the receptortranscribed by said receptor gene. In some aspects, the receptor gene isa cytokine receptor gene. In specific aspects, the cytokine receptorgene is Interleukin-2 receptor α (IL-2Rα). In particular aspects, theagonist is Interleukin-2 (IL-2). In certain aspects, the disease iscancer. In further aspects, the receptor gene is transferred into the atleast one cell via adoptive cell therapy. In some aspects, the treatmentdoes not require lymphodepletion. In other aspects, the at least onecell is a donor T cell. In certain aspects, a treatment method of theembodiments may require lower amounts of lymphodepletion relative tocurrently used clinical protocols for adoptive cell transfer.Furthermore, in some aspects, treatment methods of the embodiments cancomprise low dose administration of a cytokine ligand (followingadoptive transfer of cells). In particular, because cells of theembodiments have increased activity of a cytokine receptor (e.g.,IL-2R), lower doses of the receptor ligand (e.g., IL-2) are effective toprovide stimulation of the transferred cells.

In an additional embodiment, there is provided a method to expandspecific populations of cells in vivo. In some aspects, patients may bedirectly injected with a vector (e.g., a retroviral vector) encoding aCAR linked to an IL-2Rα. Following injection, the patient isadministered a IL-2-based therapy and the cells transduced with thisvector will preferentially expand. Vectors for use according to theembodiments include, without limitation, a retroviral vector, alentiviral vector, adenoviral vector, an adeno-associated viral vectoror a plasmid vector (e.g., delivered by a gene gun or liposome deliverysystem). In some cases, the responding cells would be only geneticallymodified for a short period of time and in other cases the cells wouldbe permanently genetically modified. For example, the vector encodingthe CAR and/or cytokine receptor can be an episomal vector or a mRNAvector.

In a further embodiment there is provided a method for providing anenhanced immune response in a subject. For example, an immunogeniccomposition can be administered to a subject in conjunction with apharmacological agents to improve receptor or receptor subunitexpression or activity (e.g., to enhance IL-2R or IL-2Rα expression oractivity). For instance, following immunization with an antigen themammalian subject can be given pharmacological agent to improve receptoror receptor subunit expression. Examples of pharmacological agentsaccording to this embodiment include, without limitation, epigeneticdrugs targeting HDAC2 or G9a that improve the durability of IL-2Raexpression. In further aspects, a subject can be administered anIL-2-based therapy that will selectively expand those cells respondingto vaccination and with elevated IL-2Ra.

Aspects of the invention provide that genetically transferring cytokinereceptor genes, such as the high affinity IL-2Rα, into lymphocytes orother cells, will dramatically enhance sensitivity to cytokine therapy.There are multiple advantages to this approach: 1) It will not benecessary to give high amounts of IL-2, which is associated with lifethreatening toxicity, as adoptively transferred cells will respond to amuch lower dose of IL-2; 2) It may be possible to make cells IL-2responsive that are not IL-2 responsive; 3) Genetic modification ofdonor T cells with IL-2Rα allows for effective adoptive cellular therapystrategies in a lymphoreplete environment. Lymphodepletion, which,although highly toxic, is often required by patients undergoing adoptivecellular therapy in order to allow donor cells to efficiently engraft(FIG. 21c ). This is thought to be due to the ability of lymphodepletionto increase the levels of endogenous cytokines. By eliminatinglymphodepletion, patients may remain eligible for other types oftherapies, such as checkpoint inhibition therapy.

Aspects of the embodiments refer to cytokine receptor activity. As usedherein receptor activity refers to signaling from a receptor when boundto the receptor ligand. Thus, a cell having increased receptor activitycan have, for example, increased receptor expression, increased receptorexpression at the cell surface, increased affinity of the receptor forits ligand (e.g., the ability of the receptor to bind to and/or releaseligand), increased receptor stability or increased receptor half-life,all of which increase the signaling activity of the receptor in thepresence of ligand.

Aspects, of the embodiments also refer to receptor genes. As used hereinthe term receptor gene encompasses genes of all subunits of a particularreceptor (e.g., the IL-2R α, β and γ subunits).

As used herein, “essentially free,” in terms of a specified component,is used herein to mean that none of the specified component has beenpurposefully formulated into a composition and/or is present only as acontaminant or in trace amounts. The total amount of the specifiedcomponent resulting from any unintended contamination of a compositionis therefore well below 0.05%. Most preferred is a composition in whichno amount of the specified component can be detected with standardanalytical methods.

As used herein in the specification and claims, “a” or “an” may mean oneor more. As used herein in the specification and claims, when used inconjunction with the word “comprising”, the words “a” or “an” may meanone or more than one. As used herein, in the specification and claim,“another” or “a further” may mean at least a second or more.

As used herein in the specification and claims, the term “about” is usedto indicate that a value includes the inherent variation of error forthe device, the method being employed to determine the value, or thevariation that exists among the study subjects.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating certain embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIGS. 1a-1e —IL-2/mAb but not IL-15/sIL-15Rα complexes induce potenteffector T cell responses in tumor-bearing mice. (a) Treatment schemefor B6 mice injected s.c. with B16 melanoma tumor cells 7 days prior tothe adoptive transfer of 3×10⁶ pmel-1 Tc1 cells. Mice were then treatedwith hIL-2/mAb (clone 5355) or hIL-15/sIL-15Rα complexes. (b) Tumorvolume from ‘a’ (n=9/group); each line represents one mouse. (*) Basedon a log-rank test and time to sacrifice (at 400 mm²) for analysis, micetreated with IL-2/mAb complexes had significantly improved outcomesversus each other condition (p<0.001 for each comparison). The averagetumor areas when treatment was initiated ranged between 15-20 mm²between the 4 groups. (c) The frequency of donor Tc1 cells in the bloodof mice (n=4/group) treated as in ‘a’ but in the absence of tumor. Eachpoint represents the average and bars indicate standard error. (d) Thefrequency of donor OT-I Tc1 cells in the blood of mice (n=5/group)treated with mIL-2/mAb_(CD122) (clone S4B6) or mIL-2/mAb_(CD25) (clone1A12). Each point represents the average and bars indicate standarderror. (e) The frequency of donor polyclonal T cells in the blood ofmice (n=5/group) treated with hIL-2/mAb (clone 5355) complexes orvehicle alone. Each point represents the average and bars indicatestandard error. For c-e, (**) indicates a significant difference(p<0.001) between indicated and other conditions. Random effects linearregression was used for modeling data and calculating p-values comparingconditions. All results are representative of at least 2 independentexperiments.

FIGS. 2a-2h —IL-2Rα mediates sustained signaling in effector CD8⁺ Tcells following withdrawal of IL-2. (a) Diagram of the standard cytokineassay in which effector cells are assayed after incubation with titratedcytokine. (b,c) Levels of pSTAT5 in Tc1 and Tc0 cells that were culturedwith increasing amounts of mIL-2 or mIL-15 for 1 hour. (d) As in ‘b’,except Tc1 cells were incubated as indicated for up to 2 hours with 200ng/ml of cytokine and assayed for pSTAT5. (e) Diagram of the cytokinepulse assay in which effector cells are incubated with saturatingamounts of cytokine (200 ng/ml). Cells are then washed thoroughly,recultured at 37° C. without additional cytokine, and assayed forpSTAT5. (f) Levels of pSTAT5 in Tc1 cells that were pulsed with mIL-2with or without anti-IL-2Rα mAb (PC61 clone) for 1 hour, washed, andrecultured at 37° C. for the times indicated. (g,h) Levels of pSTAT5 inpolyclonal effector T cells from wildtype (IL-2Rα^(+/+)) or IL-2Rα^(+/−)mice that were pulsed for 1 hour with mIL-2 or mIL-15, and assayed asdescribed in ‘e’. Except for ‘g’ and ‘h’, all effector cells weregenerated from pmel-1 mice. All results are representative of at least 3independent experiments.

FIGS. 3a-3g —IL-2Rα facilitates sustained IL-2 signaling throughcreation of an extracellular reservoir and recycling. (a) Presence ofIL-2 on the surface of polyclonal T cells depends on IL-2Rα. Polyclonaleffector CD8⁺ T cells were pulsed for 2 hours with or without mIL-2.Prior to (and during) the pulse, T cells were incubated with anti-IL-2RαmAb (PC61). Cells were then washed and stained for surface IL-2. (b)Time course of surface IL-2 on polyclonal T cells after reculture at 37°C. (c) Levels of pSTAT5 in Tc1 cells that were pulsed with IL-2, washed,and recultured at 37° C. with or without anti-IL-2 mAb (clone S4B6 or1A12). (d) Recycling of IL-2 on effector T cells. Pmel-1 Tc1 cells wereincubated with hIL-2 or mIL-2 at 37° C. for 2 hours. As indicated, cellswere then acid washed and recultured at 37° C. for 90 minutes in thepresence of anti-hIL-2 mAb conjugated to Alexa647. Cells were thenwashed, fixed, and assayed by flow cytometry. (e) Recycling of IL-2 onpulsed cells while mixed with non-pulsed cells. Pmel-1 Tc1 cells werepulsed with hIL-2 at either 4° C. or 37° C. for 2 hours, and then acidwashed. Cells were then mixed with non-pulsed CFSE-labeled Tc1 cells.The mixed cells were recultured at 37° C. for 45 minutes in the presenceof anti-hIL-2 mAb conjugated to Alexa647. Cells were then washed, fixed,and assayed by flow cytometry. (0 Internalized IL-2 leads to sustainedpSTAT5 signaling. Pmel-1 Tc1 cells were pulsed with hIL-2 at either 4°C. or 37° C. for 2 hours, and then acid washed. Cells were thenrecultured at 37° C. and assayed for pSTAT5. (g). Subcellularlocalization of hIL-2 and IL-2Rα (upper panel). Pmel-1 Tc1 cells werepulsed with hIL-2 (or media alone) for 1 hour at 37° C., and stained forhIL-2 and IL-2Rα. Cells were then imaged by confocal microscopy.Subcellular localization of hIL-2 and Rab5 (lower panel). As describedfor the upper panel, except cells were stained for Rab5. Results arerepresentative of 3 independent experiments.

FIGS. 4a-4c —IL-2Rα on donor T cells is critical for persistence inlymphoreplete but not lymphodepleted hosts. (a) Wildtype andIL-2Rα^(+/−) effector CD8⁺ T cells show similar persistence with orwithout IL-2 therapy. Effector T cells from wildtype and IL-2Rα^(+/−)mice were activated, mixed, and injected into recipient mice(n=3-5/group). Mice received injections of hIL-2/mAb (clone 5355)complexes, hIL-15/sIL-15Rα complexes, or vehicle alone. The proportionof IL-2Rα^(+/−) T cells among all donor T cells in the spleen wasdetermined pre- and post-transfer. Each triangle represents one mouseand the bars indicate the mean. (b) The total number of donor T cellsper spleen for the experiment shown in ‘a’. The bars indicate the mean.The symbol (**) indicates a significant difference (p<0.001) betweenindicated conditions. (c) The total number of donor T cells in thespleen for the experiment shown in ‘a’. Mice (n=5/group) were giventotal body irradiation (TBI, 600 rad) one day prior to adoptive transferof 10⁷ Tc1 (pmel-1) cells, and then treated with hIL-2/mAb (clone 5355)or hIL-15/sIL-15Rα complexes. The frequency of donor T cells in theblood of mice was determined at the indicated time points. Each pointrepresents the average and bars indicate standard error. The symbol (**)indicates a significant difference (p<0.001) between control andindicated conditions. All results are representative of 2 independentexperiments.

FIGS. 5a-5b —IL-2/mAb complexes selectively enhance the persistence ofdonor T cells. B6 mice (n=6-7/group) were injected intravenously with8×10⁶ Tc1 pmel-1 CD8₊ T cells. On days 0, 2, 4, 6, as indicated, micereceived (i.p.) either hIL-2/mAb (clone 5355) or hIL-15/sIL-15Rαcomplexes. (a) The frequency of donor CD8₊ T cells in the spleens, lymphnodes and liver were determined on day 8. Each triangle represents onemouse and the bar indicates the mean. The symbol (**) indicates asignificant difference (p<0.001) between indicated conditions. (b)Splenocytes from mice treated as in ‘A’ were stimulated with or withouthgp100₂₅₋₃₃ peptide for 5 hours. The frequency of donor T cells positivefor both IFNγ and TNFα was determined by flow cytometry. Results arerepresentative of 2 independent experiments.

FIG. 6—In the absence of donor T cells, hIL-2/mAb and IL-15/sIL-15Rαcomplexes mediate comparable anti-tumor immunity. B6 mice (n=8/group)were injected with B16 tumor cells. The next day, mice were given i.p.injections as indicated of either hIL-2/mAb (clone 5355) orhIL-15/sIL-15Rα complexes for 7 days. (For hIL-2/mAb we used 1 μg ofcytokine and 5 μg of antibody, and for hIL-15/sIL-15Rα we used 0.5 μg ofcytokine and 2.3 μg of soluble receptor per injection.) Tumors weremeasured in a blinded fashion twice a week. Each line is representativeof one mouse. IL-2/mAb and IL-15/sIL-15Rα complexes significantlyincreased the time to sacrifice versus the control condition (log-ranktest, <0.05). Results are representative of 2 independent experiments.

FIG. 7—Treatment with IL-2/mAb, IL-2/mAb_(CD25), and IL-15/sIL-15Rαcomplexes induces differential expansion of CD8₊ memory-phenotype Tcells, NK cells, and T regulatory cells. B6 mice (n=5/group) wereinjected with hIL-2/mAb (clone 5355), hIL-2/mAb_(CD25) (clone 5344.111),or hIL-15/IL15Rα complexes on days 0, 2, 4, and 6. Spleens wereharvested on day 8 and stained for T regulatory cells (CD4₊CD25₊FOXP3₊),memory-phenotype (MP) CD8 T cells (CD8₊CD44^(hi)), and NK cells(NK1.1₊TCRβ-B220⁻). Mice also received adoptive transfer of Tc1 cells(data not shown). (**, p<0.001 or *, p=0.008) indicates a significantdifference between indicated conditions and control. Data isrepresentative of two independent experiments.

FIG. 8—Tc1 but not Tc0 effector CD8₊ T cells show preferentialresponsiveness to IL-2/mAb complexes. B6 mice (n=6-7/group) wereinjected intravenously with 8×10₆ Tc1 or Tc0 pmel-1 CD8₊ T cells. Ondays 0, 2, 4, 6, as indicated, mice received (i.p.) either hIL-2/mAb(clone 5355) or hIL-15/sIL-15Rα complexes. The top graph shows thefrequency of donor CD8₊ T cells in the spleens on day 8. The bottomgraph shows the absolute number of donor T cells on day 8. Each trianglerepresents one mouse and the bar indicates the mean. These data are fromthe same experiment shown in supplemental FIG. 1. Values werelog-transformed prior to comparison of means by two-sample t-tests.

FIGS. 9a-9b —Blockade of IL-2Rα has minimal impact on Tc1 cells inresponse to titrated IL-2(a) or IL-15(b). Using a standard cytokineresponsiveness assay, Tc1 cells from pmel-1 mice were incubated withtitrated amounts of mIL-2 or mIL-15 for 30 minutes and assayed forpSTAT5. As indicated, anti-IL-2Rα mAb (PC61) was added at 5 μg/ml.Results are representative of 3 similar experiments.

FIGS. 10a-10b —Tc1 effector CD8₊ T cells exhibit comparable functionalsensitivity to IL-2 and IL-15 in vitro. Tc1 CD8₊ T cells generated frompmel-1 TCR transgenic mice were plated with either IL-2 or IL-15. After48 hours, the frequency of proliferating (a) and viable (b) cells wasassayed by Ki67 staining and propidium iodide (PI) exclusion,respectively. Cells were then analyzed by flow cytometry.

FIGS. 11a-11c -Tc1 effector CD8₊ T cells pulsed with IL-2 mediatesustained cytokine signaling. (a) In the cytokine pulse assay, Tc1 orTc0 effector CD8₊ T cells were incubated overnight at 37° C. with mIL-2(200 ng/ml), mIL-15 (200 ng/ml), or without cytokine. Cells were thenwashed thoroughly, recultured at 37° C. without additional cytokine, andassayed for phosphorylation of STAT5. The frequency of cells stainingpositive for pSTAT5 are shown for (b) Tc0 and (c) Tc1 cells. Results arerepresentative of 3 independent experiments.

FIG. 12—IL-2 mediated sustained cytokine signaling is IL-2Rα-dependentin 11 independent experiments. Tc1 cells from pmel-1 mice were pulsedwith mIL-2 with or without anti-IL-2Rα mAb (PC61 clone) for 90 minutes,then washed and recultured at 37° C. Cells were harvested at the timesindicated and stained for pSTAT5. Each symbol represents one of 11independent experiments.

FIGS. 13a-13b -Tc1 effector CD8₊ T cells pulsed with IL-2 exhibitIL-2Rα-dependent proliferation after cytokine withdrawal. (a) Tc1 cellsfrom pmel-1 TCR transgenic mice were pre-incubated as indicated withanti-IL-2Rα mAb (PC61) or isotype control antibody for 15 minutes. Then,mIL-2 or mIL-15 was added for 2 hours at 37° C. Cells were then washedthree times and resuspended in culture media without cytokine for 18hours. During the last hour of culture, BrdU was added. Cells were thenstained for BrdU and CD8, and analyzed by flow cytometry. (b) Thefrequency of CD8₊ T cells positive for BrdU staining in cytokine-treatedcultures is indicated by the black line and the number in the upperright quadrant. Control cultures without cytokine are indicated by theshaded histogram. Results are representative of 3 independentexperiments.

FIG. 14—Human IL-2 mediates sustained cytokine signaling on mouse Teleffector CD8₊ T cells. Tc1 cells from pmel-1 mice were pulsed with hIL-2with or without anti-IL-2Rα mAb (PC61 clone) for 90 minutes, then washedand recultured at 37° C. Cells were harvested at the times indicated andstained for pSTAT5. Results are representative of 5 independentexperiments.

FIGS. 15a-15b -Human effector CD8₊ T cells pulsed with IL-2 mediatesustained IL-2Rα-dependent signaling. (a) Human PBMCs activated withplate-bound anti-CD3 mAb for 3 days were pulsed with either hIL-2 orhIL-15 at 37° C. for one hour. Effector cells were then washed to removeunbound cytokine and recultured in media without cytokine at 37° C. Atthe indicated times, cells were fixed and stained for CD8 and pSTAT5.The percentage indicates the frequency of CD8₊ T cells staining positivefor pSTAT5. (b) Human PBMCs from two healthy adult donors were activatedfor 2 days with plate-bound anti-CD3 mAb. Effector cells were thenpulsed with hIL-2 in the absence or presence of an anti-IL-2Rα pAb (R&Dsystems, AB-223-NA) for two hours. pSTAT5 was assessed in these cells atthe indicated times in a manner similar to ‘a’. For ‘a & b’, similarresults were obtained with CD8₊ T cells derived from 4 healthy adultdonors.

FIGS. 16a-16b -Human IL-2/mAb (clone 5355), but not mouseIL-2/mAb_(CD122) (clone S4B6) complexes, are permissive toIL-2Rα-dependent sustained signaling in vitro. (a) Tc1 cells from pmel-1TCR transgenic mice were incubated with hIL-2 with or without excessanti-hIL-2 mAb (clone 5355, 10 μg/ml) to generate hIL-2/mAb in vitro. Inreplicate wells, anti-IL-2Rα mAb (clone PC61) was added during theincubation step to block IL-2Rα-dependent signaling. Cells were thenwashed and recultured at 37° C. for the time indicated. Phosphorylationof STAT5 was assessed at the indicated time points by flow cytometry.(b) As in ‘a’, except mouse IL-2 and anti-mIL-2 mAb (clone S4B6, 10μg/ml) were used to generate mL-2/mAb_(CD122) in vitro. Results arerepresentative of two independent experiments.

FIG. 17—Antibodies for mouse and human IL-2 are species-specific. Tc1cells from pmel-1 TCR transgenic mice were pulsed with mIL-2 (200ng/ml), hIL-2 (200 ng/ml), or mIFNγ (200 ng/ml) for 45 minutes. Cellswere then stained with either anti-mIL-2 mAb or anti-hIL-2 mAb directlyconjugated to Alexa647 and analyzed by flow cytometry. Data arerepresentative of 3 independent experiments.

FIGS. 18a-18b —Detection of hIL-2 by confocal microscopy isspecies-specific and dependent on pulsing cells with cytokine at 37° C.(a) Tc1 cells generated from pmel-1 TCR transgenic mice were pulsed withhIL-2 (200 ng/ml), mIL-2 (200 ng/ml), or media alone (control) for 90minutes at 37° C. Cells were then fixed, permeabilized, and stained withanti-hIL-2 mAb prior to being mounted onto slides. IL-2 staining inconfocal images is represented as a red pseudocolor. (b) As in ‘a’,except cells were pulsed with hIL-2 at 4° C. or 37° C. All results arerepresentative of at least two independent experiments.

FIG. 19—Detection of mIL-2 by confocal microscopy. (a) Tc1 cellsgenerated from pmel-1 TCR transgenic mice were pulsed with mIL-2 (200ng/ml) or media alone (control) for 90 minutes at 37° C. Cells were thenfixed, permeabilized, and stained with anti-mIL-2 mAb prior to beingmounted onto slides. Results are representative of two independentexperiments.

FIGS. 20a-20c -Colocalization of hIL-2 with EEA-1 and LAMP-1 by confocalmicroscopy. Tc1 cells from pmel-1 TCR transgenic mice were pulsed withhIL-2 for 90 minutes at 37° C., and stained for hIL-2, EEA1, or LAMP-1.Cells were then imaged by confocal microscopy to determine thesubcellular localization of hIL-2 relative to EEA-1 and LAMP-1. Ninerepresentative images for EEAVIL-2 and LAMP-1/IL-2 were taken, andscored blindly by three independent observers. The percentcolocalization was determined by counting the sum of IL-2 directlycolocalizing (yellow) versus IL-2 colocalizing (yellow) plus IL-2 alone(green). Each solid line below denotes readings by one rater of %colocalization of EEA-1 and LAMP-1. “X” values and dashed line indicateestimated colocalization from regression model, adjusting for ratervariability. Mean difference in LAMP-1 and EEA-1 colocalization isstatistically significant (p=0.010).

FIGS. 21a-21c —In lymphodepleted mice, IL-15/sIL-15Rα and hIL-2/mAbmediate comparable engraftment of Tc1 effector CD8₊ T cells. (a) Diagramdepicting the ability of IL-2 to preferentially engage IL-2Rα^(hi) donorT cells, while IL-15 requires removal of host cells for equivalentactivity on donor T cells. (b) Mice (n=5/group) were treated without(top) or with (bottom) total body irradiation (TBI, 600 rad) one dayprior to adoptive transfer of 10₇ pmel-1 Tc1 cells. Then on days 0, 2,4, and 6, mice were treated with hIL-2/mAb (clone 5355) orhIL-15/sIL-15Rα complexes. Spleens were harvested on day 8. Eachtriangle represents one mouse and the bar indicates the mean. (**)indicates a significant difference (p<0.001) between control andindicated conditions. Results are representative of 2 independentexperiments. (c) A schematic showing the effect of IL-2 of adoptive celltransfer (ACT) therapy with ACT only, ACT plus lymphodepletion and ACTusing cells having increased IL-2R activity (IL-2Rα^(hi)).

FIG. 22—An example nucleic acid vector of the embodiments. Shown is aretroviral vector encoding T-cell receptor (TCR) genes (alpha and beta)and an IL-2Rα(CD25) molecule. The TCR could be specific for, as anexample, a tumor antigen such as MART-1 or tyrosinase. In this case, thevector could be used to genetically modify T cells, which will then betransferred into a cancer patient. A patient having cells comprisingsuch a vector will be able to respond much more efficiently toIL-2-based therapies.

FIG. 23—Low-dose IL-2 leads to preferential expansion of adoptivelytransferred donor tumor-reactive T cells by engagement of IL-2Rα. B6mice were injected with 250,000 B16-F1 tumor cells (s.c.). Eight dayslater, mice were adoptively transferred with 3×10⁶ tumor-reactiveactivated T cells (pmel-1) conditioned with IL-12 to induce high levelsof IL-2Rα. On the day of adoptive T cell transfer, 2 days later, and 4days later, mice were treated with hIL-2 (1.5 ug), hIL-2/mAb complexes(1.5 ug hIL-2 and 7.5 ug anti-hIL-2 mAb (MAB602)), or hIL-15/sIL-15Rα-Fccomplexes (0.5 ug hIL-15+2.3 ug sIL-15Rα-Fc). On day 6 after adoptivetransfer, mice were bled and the frequency of donor T cells(CD8⁺Thy1.1⁺) in the peripheral blood was determined. Each trianglerepresents an individual mouse and the bar indicates the mean. Thenumber in parenthesis and in red indicates the frequency of donor Tcells in the blood.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS I. The Present Embodiments

In some aspects, methods detailed herein concern adoptively transferringlymphocytes that have been modified to express elevated levels ofcytokine receptor genes or cytokine receptor subunit genes. For example,tumor-reactive T cells can be modified to express IL-2Rα. Upon adoptivetransfer, these cells will have enhanced ability to respond to theexogenous ligand. Thus, in this example, tumor-reactive T cells willhave enhance ability to respond to exogenously administered IL-2 or asimilar IL-2-based reagent. Cells responding to cytokine havesignificant advantage for growing and mediating effector functions suchas killing tumor cells. Thus, this approach may allow clinicians toadminister adoptive cellular therapy without having to preconditionpatients with chemotherapy or radiation to deplete host lymphocyteswhich normally compete for cytokine. Moreover, the instant methodsprovide the ability to genetically modify lymphocytes in vivo andprovide them cytokine receptor genes or cytokine receptor subunit genes.For example, a subject may be injected with a retroviral vectorcontaining IL-2Rα and a CD19-reactive CAR. Cells modified with such avector would be very responsive to IL-2-based therapy, and therefore,this method would provide an effective means for expanding such cells.Methods, such those described above, have application for cancer therapyas well as for the treatment of other disease. For example, T regulatorycells might be genetically modified with IL-2Rα, and exhibit improvedresponsiveness to IL-2 therapy, and thus, this approach could haveapplication for the treatment of autoimmune disease.

As noted above, in some specific examples, methods of the embodimentscan be used to produce cells having enhanced responsiveness to IL-2.Administration of IL-2 is a critical component of many T cell-basedstrategies for cancer therapy. However, IL-2 has a short half-life anddose limiting toxicity. Furthermore, as IL-2 selectively expands Tregulatory cells, it has been proposed that IL-15-based therapies maymore effectively support adoptively transferred effector T cells. Thefindings here show that genetically transferring cytokine receptorgenes, such as IL-2Rα, into lymphocytes or other cells dramaticallyenhances sensitivity to cytokine therapy. This approach is easilyadopted for other cytokines or injectable protein therapeutics dependenton receptor expression. For example, the technique could be used togenetically transfer IL-15Rα to modulate lymphocyte responsiveness toIL-15. It is also possible to create chimeric or novel receptors combingdifferent ligand binding and cell signaling properties. It is alsopossible to genetically add receptors in vivo through novel genetransfer techniques. Alternatively, in some instances, receptor genesmay be introduced in a transient method (such as RNA electroporation),so that the impact is not long lasting.

In some embodiments, the treatment entails genetically modifyinglymphocytes with other proteins that enhance cytokine receptor geneexpression. This could include the transfer of transcription factorsthat lead to up-regulation of cytokine receptors or enhance the cellularmachinery necessary for cytokine responsiveness.

In another embodiment, receptor expression is modulated in other waysthan outlined above. For example, modulation of the levels of IL-2Rβ andIL-2Rγ, either individually, together, or with or without modulation ofIL-2Rα, may be done. As part of this, modulation may be done byincreasing the expression of these receptors or by inhibiting theexpression of the endogenous receptor(s). For example, geneticmodification of IL-2Rα and Il-2Rβ may be necessary for optimalresponsiveness to IL-2.

As an additional aspect of the embodiments, mutant or altered versionsof IL-2 may be used. For example, a mutant recombinant IL-2 molecule maybe used to enhance binding to IL-2Rα. In some cases a mutant IL-2 mayalso have altered affinity for IL-2Rα dependent on pH (and thus, mayundergo differential intracellular trafficking). The treatment may usean IL-2 molecule fused to another protein such as IgG. These alteredIL-2 molecules may provide for improved IL-2 responsiveness and act inan additive or synergistic manner to the genetic modification of T cellsas proposed above.

In another embodiment, altered receptor molecules are designed. Forexample, a version of IL-2Rα with improved sensitivity to IL-2 may bemore effective upon genetic modification of lymphocytes.

In another embodiment, genetic constructs including long terminalrepeats (LTR) linking T-cell receptor (TCR) or chimeric antigen receptor(CAR) genes to cytokine receptor subunits are created. For example, TCRαor TCRβ is linked to IL-2Rα, where the TCR genes are reactive against amelanoma tumor antigen. The genetic construct used could be a retroviralvector, lentiviral vector, or any other means of genetically modifying Tcells using DNA or RNA. In addition to these genetic elements, othergenes may be linked to this construct such as a selectable marker (CD34or GFP) or a suicide gene to allow killing of the adoptively transferredcell population.

In some embodiments, this approach is used to modify other cells, suchas specific lympohcyte subusets (such as T regulatory cells, Tc1 cells,or Th17 cells), or completely different classes of lympohcytes such asnatural killer cells.

As detailed above, in some embodiments a method of treating a patient isprovided. For example, in the case of a patient with metastaticmelanoma, who seeks treatment with adoptive cellular therapy, tumorinfiltrating lympohcytes (TIL) can be isolated from this patient andexpanded to later numbers for adoptive transfer. During this process,the TIL can be genetically modified with a retroviral vector encoding anIL-2R gene. While normally, such a patient might be givenlymphodepleting non-myeloablative chemotherapy with cyclophosphamide andfludarabine, with IL-2Rα-modified TIL, this patient may not require suchchemotherapy to enhance TIL efficacy or may require a lower dose ofchemotherapy. In this situation, the patient may be given low dose IL-2therapy. Alternatively, the patient could be given another IL-2-basedmolecule such as an IL-2 fusion protein.

In a further example, a patient with B cell-derived malignancy, whoseeks treatment with adoptive cellular therapy, can have peripheralblood genetically modified with a CAR vector also containing an IL-2Rαgene. In some cases, there may also be a suicide gene in this vector.The patient can be treated with the CAR-IL-2Rα-modified T cells and lowdose IL-2. In this case, the patient may not require chemotherapy tosuppress the host immune cells.

In another example, a patient with metastatic melanoma who seekstreatment with adoptive cellular therapy, can have tumor infiltratinglympohcytes (TIL) isolated and expanded to sufficient numbers foradoptive transfer. During this process, the TIL can be geneticallymodified with a retroviral vector encoding IL-12 receptor (IL-12Rβ1and/or IL-12Rβ2). In this situation, very low doses of IL-12 may augmentability of TIL to mediate anti-tumor efficacy. In this case, IL-12 canbe given at lower doses and may not be toxic to the patient. Thisexample could be applied to any cytokine, ligand, or protein therapywhere efficacy is impacted by dose limiting toxicity.

In still another example, a patient may require a bone marrowtransplant. In this case, the bone marrow cells may be geneticallymodified with a vector encoding IL-2Rα, GM-CSF receptor (GM-CSF receptorα and βc), or G-CSF receptor (GCSF-receptor). In this case, the patientmay be given GM-CSF or G-CSF ligand, to induce improved reconstitutionof the bone marrow graft or IL-2 to selectively reconstitute T cellswithin the graft. This technology could be used with any cytokine orligand receptor system.

Still another example of method of treatment in accordance with theembodiments concerns a patient with metastatic melanoma who seekstreatment with adoptive cellular therapy. Tumor infiltrating lympohcytes(TIL) can be isolated from this patient and expanded to sufficientnumbers for adoptive transfer. During this process, the TIL can begenetically modified with a retroviral vector encoding a mutated IL-2Rαgene. The mutation may eliminate potential ribosylation sites, andtherefore make the IL-2Rα more responsive to IL-2 therapy.Alternatively, the IL-2Rα molecule may be mutated so that theintracellular signaling domain from another receptor subunit orcostimulatory molecule is engineered into the intracellular portion ofIL-2Rα. In this case, the IL-2Rα may improve T cell function in novelways. For this patient, while normally, he or she may be givenlymphodepleting non-myeloablative chemotherapy with cyclophosphamide andfludarabine, with IL-2Rα-modified TIL, this patient may not require suchchemotherapy to enhance TIL efficacy. In this situation, the patient maybe given low dose IL-2 therapy. Alternatively, the patient could begiven another IL-2-based molecule such as an IL-2 fusion protein.

II. Examples

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1—Materials and Methods

Study Design.

This was a preclinical study to assess the efficacy of cytokine therapyto augment anti-tumor T cell immune responses. The inventors found thatIL-2-based therapies were more efficacious than IL-15-based therapies inthe tumor model, and thereafter, focused on understanding the mechanismof this differential response. For in vivo experiments, the numbers ofmice are outlined in the figure legends. For all experiments, the numberof independent replicates is outlined in each figure legend.Randomization and blinding for tumor experiments was done as describedin the tumor methods below. Additional study design details are alsoincluded in the statistical analysis section below.

Recombinant Proteins and Antibodies.

Human (h) IL-15, hIL-2, and anti-hCD3 mAb (clone OKT3) were kindlyprovided by the NCI Biological Resources Branch Preclinical Repository.Mouse (m) IL-2, mIL-12, and mIL-15 were purchased from ShenandoahBiotechnology. Recombinant sIL-15Ra-Fc (551-MR-100) and anti-hIL-2 mAb(clone 5355) were purchased from R&D systems. Anti-hIL-2 mAb_(CD25)(clone 5344.111) was obtained from BD Bioscience. Anti-mIL-2 mAb_(CD122)(clone S4B6) and anti-IL-2Rα (clone PC61) were obtained from Bioxcell.Anti-mIL-2 mAb_(CD25) (clone JES6-1A12), anti-mCD3 mAb (clone 145-2C11),and anti-mCD28 mAb (clone 37.51) were obtained from the UCSF monoclonalantibody core. Antibodies used for flow cytometric and confocal analysisare described below.

Mice and Tumor Cells.

C57BL/6 (B6), B6.PL (Thy1.1), B6(CD45.1), pmel-1 TCR transgenic, andOT-I TCR transgenic mice were purchased from Jackson Laboratory. Allanimals were housed under specific pathogen-free conditions inaccordance with institutional and federal guidelines. For tumorexperiments, B16-F1 cells were obtained from ATCC.

T Cell Cultures.

Mouse Tc1 and Tc0 cells were generated from pmel-1 and OT-I TCRtransgenic mice as previously described (Rubinstein et al., 2012).Briefly, splenocytes were cultured for three days with relevant peptide(for pmel-1, hgp100₂₅₋₃₃ peptide (KVPRNQDWL) and for OT-I, OVA₂₅₇₋₂₆₄peptide (SIINFEKL)) and cultured with (Tc1) or without (Tc0) mIL-12(long/ml). Polyclonal mouse T cells were generated by culturing B6splenocytes for three days with plate-bound anti-CD3 mAb (clone145-2C11, 1 ug/ml) unless otherwise stated. Activated human T cells weregenerated by culturing de-identified human PBMCs (Research BloodComponents) from healthy adult donors for two or three days withplate-bound anti-CD3 mAb (clone OKT3, 1 ug/ml).

Tumor and Persistence Studies in Mice.

For tumor experiments, B6 mice were challenged subcutaneously with2.5×10⁵ B16-F1 tumor cells. Prior to randomizing mice to treatmentgroups, some mice were excluded due to abnormal tumor growth. Asindicated, mice were treated by adoptive transfer of activated T cells(Tc1 or Tc0) by intravenous tail vein injection. Cytokine complexes wereadministered by intraperitoneal injection on days 0, 2, 4, and 6 afteradoptive transfer unless otherwise indicated. Cytokine complexes usedinclude: hIL-15/sIL-15Rα, hIL-15 (0.5 ug)/sIL-15Ra-Fc (2.3 ug);hIL-2/mAb, hIL-2 (1.5 ug)/anti-IL-2 mAb (7.5 ug, clone 5355);hIL-2/mAb_(CD25), hIL-2 (1.5 ug)/anti-IL-2 mAb (7.5 ug, clone 5344.111);mIL-2/mAb_(CD122), mIL-2 (1.5 ug)/anti-IL-2 mAb (7.5 ug, clone S4B6);and mIL-2/mAb_(CD25), mIL-2 (1.5 ug)/anti-IL-2 mAb (7.5 ug, cloneJES6-1A12). Tumor growth was measured by caliper every 2-4 days bypersonnel blinded to the treatment regimen. Tumor surface area (mm²) wascalculated as length×width. Mice were sacrificed when tumors reached 400mm². For persistence studies, mice received adoptive transfer ofactivated T cells (Tc1 or Tc0). Peripheral blood lymphocytes orindicated organs were stained for CD8 and either Thy1.1 or CD45.1 toidentify donor T cells. In experiments with a mixed transfer, theinventors used effector T cells from wildtype (Thy1.1) and IL-2Rα^(+/−)(Thy1.2) mice that were activated with plate-bound anti-CD3/anti-CD28mAb, mixed, and transferred into B6(CD45.1) mice.

Where indicated, mice also received total body irradiation (600 rad) oneday prior to adoptive T cell transfer. In all adoptive transferexperiments, donor and recipient mice were gender-matched and were 6-12weeks of age. All animals were housed under specific pathogen-freeconditions in accordance with institutional and federal guidelines.

Flow Cytometry.

Flow cytometry analysis was performed as previously described (17). Theantibodies used in this study include CD8 (53-6.7), CD25 (PC61), CD45.1(A20), IFNγ (XMG1.2), STAT5 pY694 (47/Stat5(pY694)), Thy1.1 (A20), andTNFα (TN3-19.12). These were purchased from BD Bioscience, Biolegend(San Diego, Calif.), and eBioscience (San Diego, Calif.). For analysisof phosphorylation of STAT5, the inventors followed the manufacturer'sprotocol using Lyse/Fix and PermIII buffer (BD Bioscience). To examinecellular proliferation, cells were fixed and permeabilized according tothe manufacturer's protocol for Cytofix/Cytoperm (BD Bioscience) andstained with anti-Ki67 mAb (SolA15, eBioscience). Alternatively, BrdU(10 μm) was added one hour prior to harvest, and cells were analyzed forBrdU incorporation as previously described (Rubinstein et al., 2008).For Foxp3 staining, the inventors followed the protocol outlined in theFoxp3 kit (eBioscience). Flow cytometry was performed on BD LSRII and BDFACSAccuri. Data were analyzed using FlowJo software (TreeStar). In allexperiments, initial gating of live cells was performed using forwardscatter and side scatter parameters, and cells were then gated on livelymphocytes. Isotype and fluorescence minus one (FMO) controls wereperformed as required. For experiments assessing IL-2, the inventorsalways included control conditions without IL-2 pulsing.

In Vitro Experiments.

For functional assays, Tc1 or Tc0 cells were incubated with cytokinesand assayed for pSTAT5, Ki67, BrdU, or propidium iodide exclusion. Forpulse assays, cells were incubated with or without cytokine at 200 ng/mlat either 4° C. or 37° C. for 90 minutes unless otherwise indicated.Cells were then washed at least three times, replated without cytokine,and assayed for pSTAT5. When added during the pulse step, anti-IL-2RαmAb was added 15 minutes prior to cytokine addition. Acid wash wasperformed by washing cells twice for 2 minutes at 4° C. with an acidwash buffer consisting of complete media adjusted to pH3.5 or pH3.75with 1N HCl. For analysis of recycling of IL-2 to the cell surface, acidwashed cells were replated in media at 37° C. for the indicated amountof time with anti-IL-2 mAb conjugated to Alexa647. To assess IFNγ andTNFα production, the inventors added hgp100₂₅₋₃₃ (1 ug/ml) or PMA (50ng/mL) and ionomycin (1 μM) to splenocytes for 6 hours in the presenceof brefeldin A (GolgiStop, BD Bioscience).

Confocal Microscopy.

Tc1 cells were incubated with hIL-2 (200 ng/ml), mIL-2 (200 ng/ml), orno cytokine, for 1 hour at either 4° C. or 37° C. unless otherwisestated. Cells were washed, fixed, and permeabilized using theCytofix/Cytoperm protocol. To determine the subcellular localization ofinternalized IL-2 by confocal microscopy, cells were stained withanti-hIL-2 mAb and either anti-IL-2Rα pAb (R&D systems), anti-Rab5 mAb(C8B1, Cell Signaling), anti-LAMP1 mAb (1D4B, company), or anti-EEA1 mAb(C45B10, Cell Signaling). To detect anti-IL-2Rα, the inventors used ananti-goat IgG conjugated to Alexa488 (R&D systems). To detect EEA-1 andRab5, the inventors used an anti-rabbit IgG conjugated to Alexa488(F(ab′)2 fragment, Cell Signaling). After washing, cells weretransferred to SuperFrost microscope slides via cytospin.Immunofluorescence staining was visualized with a confocal microscope(Olympus Fluoview FV10i laser scanning confocal microscope system,Olympus) using a 60× water immersion objective (1.2 NA). Image analysiswas performed using the FV10-ASW 1.7 software. In all images, IL-2staining is presented as a red pseudocolor. In all experiments, cellspulsed without IL-2 were used as the primary control.

Statistical Analyses.

Before analysis, graphical displays were made of all data vs. conditionsto identify the need for transformations to adhere to model assumptions.For experiments comparing outcomes at a fixed point in time, logtransforms were taken and comparisons of means performed usingtwo-sample t-tests or linear regression (depending on the number ofconditions). Where appropriate, t-tests assumed unequal variance acrossconditions. Comparisons of conditions where mice were followed over timewere made at individual timepoints based on random effects linearregression models (with random effects to account for correlation ofdata from the same mouse over repeated measures) with the outcome (e.g.% T-cells) log-transformed. Graphical displays were used to assessappropriateness of transformation. Residual plots were inspected toassess assumptions of linear regression models. Time to sacrifice wascompared across groups using log-rank tests. Time to sacrifice wascompared across groups using log-rank tests. Percent colocalization wascompared with log(percent) as the outcome (due to skewness) and maineffects of LAMP-1 (vs. EEA-1) and rater. The LAMP-1 effect was evaluatedbased on the Wald test of the regression coefficient. Model results wereexponentiated to provide point estimates for LAMP-1 and EEA-1colocalization. In the interest of addressing the hypotheses and notover-testing, the inventors did not perform hypothesis tests for everypossible comparison in each figure. Where comparisons were insignificant(p>0.05) it is stated in the text; where tests were significant, it isstated and/or indicated with asterisks in figures. P-values are reportedto two significant digits, except when the p-value is less than 0.001;for p-values smaller than 0.001, it is reported as ‘p<0.001’. P-valuesare not corrected for multiple comparisons. For all analyses,statistical significance was based on a two-sided a level of 0.05.Statistical analyses were performed using Stata/IC (version 12.1) and Rstatistical software.

Example 2—Results

IL-2—but not IL-15—Therapy Mediates Anti-Tumor Immunity after AdoptiveTransfer of Activated CD8⁺ T Cells.

To assess the impact of cytokine therapy on adoptively transferredeffector CD8⁺ T cells, the inventors used IL-2/anti-IL-2 mAb (IL-2/mAb)and IL-15/sIL-15Rα-Fc (IL-15/sIL-15Rα) complexes, in which the antibodyor receptor acts as a carrier molecule to improve the half-life andbiological activity of free cytokine in vivo (Rubinstein et al., 2006;Stoklasek et al., 2006; Boyman et al., 2006). To test effector T cellresponsiveness to cytokines in a clinically relevant model, B6 mice wereinjected (s.c.) with B16 melanoma tumor cells (FIG. 1A). After theestablishment of palpable tumors, unirradiated mice received activatedIL-12-conditioned T cells (Tc1) from pmel-1 TCR transgenic mice, fromwhich CD8₊ T cells recognize an endogenous B16 tumor antigen(H-2D^(b)-restricted gp100₂₅₋₃₃ peptide). The inventors have shown theseTc1 effector cells are highly efficacious against tumor inlymphodepleted mice (Rubinstein et al., 2012). For the first week afteradoptive transfer, IL-15/sIL-15Rα or IL-2/mAb (clone 5355) complexeswere administered every 48 hours. While 6 of 9 mice that receivedIL-2/mAb complexes were cured of established tumor, mice that receivedeither IL-15/sIL-15Rα complexes or no cytokine therapy showed no tumorregression (FIG. 1b ). To better understand this differential response,the inventors assessed the persistence of donor Tc1 cells in recipientsthat received treatment with IL-2/mAb complexes or IL-15/sIL-15Rαcomplexes. Independent of the presence of tumor, only IL-2/mAb complexesenhanced the persistence of effector CD8⁺ T cells in a systemic fashionacross multiple organs (FIGS. 1c and 5a ). Notably, withoutlymphodepletion or vaccination, the inventors routinely achievedsustained donor T cell frequencies of 20% or higher in the peripheralblood. Furthermore, donor Tc1 cells were equally functional acrosstreatment groups as indicated by the ability to produce IFNγ and TNFα(FIG. 5b ). Finally, as a control, the inventors found that the transferof tumor-reactive effector CD8⁺ T cells was necessary for curativetherapy. Thus, tumor-bearing mice treated with only IL-2/mAb orIL-15/sIL-15Rα complexes exhibited minimally delayed tumor growth,albeit comparable between cytokine conditions (FIG. 6).

Donor T Cell Expression of IL-2Rα is Critical for PreferentialIL-2-Mediated Responses.

The preferential response of effector CD8⁺ T cells to IL-2/mAb but notIL-15/sIL-15Rα complexes was contrary to the expectation. This responsewas not dose related as IL-2/mAb and IL-15/sIL-15Rα complexes expandedIL-2Rβγ^(hi) cells such as memory-phenotype CD8⁺ T cells and NK cells toa similar extent in vivo (FIG. 7) (Rubinstein et al., 2008). However,only IL-2/mAb complexes expanded T regulatory cells (FIG. 7), which arecharacterized by their expression of IL-2Rα. As IL-12-conditioned (Tc1)effector CD8⁺ T cells express very high levels of IL-2Rα (Rubinstein etal., 2012), the results suggested an unappreciated role for cell surfaceIL-2Rα on effector T cells in dictating responsiveness to IL-2 therapy.To formally test this, the inventors made use of two anti-IL-2 mAbs withthe ability to differentially redirect IL-2 based on lymphocyte cellsurface IL-2Rα expression. IL-2/mAb_(CD25) complexes (clone 1A12)preferentially expand IL-2Rα^(hi) lymphocytes, while IL-2/mAb_(CD122)complexes (clone S4B6) act in an IL-2Rα-independent manner (Boyman etal., 2006; Spangler et al., 2015). The inventors tested these twocomplexes in lymphoreplete mice injected with Tc1 cells. For only thisexperiment, the inventors generated Tc1 cells from another TCRtransgenic mouse, OT-I, to confirm the results with a different TCR.While IL-2/mAb_(CD122) complexes mediated a minimal increase inpersistence, IL-2/mAb_(CD25) complexes induced donor T cell levels ofgreater than 60% of total lymphocytes (FIG. 1d ). To further confirmthat this effect was dependent on IL-2Rα and not on IL-12 conditioningor selective TCR engagement, the inventors stimulated polyclonal T cellsfrom wildtype mice with plate-bound anti-CD3 mAb, a method thatgenerates IL-2Rα^(hi) effector CD8⁺ T cells. Upon adoptive transfer intolymphoreplete mice, IL-2/mAb complexes (clone 5355) greatly enhanced thepersistence of polyclonal T cells (FIG. 1e ). Finally, as an additionalcontrol, Tc0 cells, which have lower levels of surface IL-2Rα(Rubinstein et al., 2012), showed limited IL-2/mAb-driven persistence(FIG. 8).

IL-2Rα Induces Sustained IL-2 Signaling in Effector CD8⁺ T Cells afterCytokine Withdrawal.

To uncover the mechanism behind the remarkable IL-2Rα-dependentresponsiveness of effector Tc1 cells in vivo, the inventors assayed IL-2and IL-15 activity downstream of IL-2Rβγ using standard in vitro assaysquantifying phosphorylation of STAT5 (a proximal signaling event),viability, and proliferation (FIG. 2a ). In the context of STAT5phosphorylation in response to titrated cytokine, the inventors foundthat Tc1 (IL-2Rα^(hi)) cells exhibited marginally increased sensitivityto IL-2 versus IL-15 when compared to Tc0 effector cells (IL-2Rα^(med))(FIGS. 2b-2c ), which is consistent with previous findings (Lisiero etal., 2011). The addition of a blocking antibody (anti-IL-2Rα mAb, PC61clone) also showed a minimal benefit of IL-2Rα engagement on Tc1 cellsin comparison between titrated IL-2 and IL-15 (FIGS. 9a-9b ). Notably,Tc1 cells responded comparably to IL-2 and IL-15 in standard assays ofproliferation and viability (FIGS. 10a-10b ). Importantly, there was nodifference in the kinetics of STAT5 phosphorylation between cellscultured in IL-2 or IL-15 (FIG. 2d ). The mildly enhanced sensitivity ofTc1 cells to IL-2 versus IL-15 seemed unlikely to account for thedramatic difference in activity observed in vivo. Therefore, theinventors hypothesized that IL-2Rα does not simply improve cellularaffinity for IL-2, but allows for sustained IL-2 signaling after a Tcell transitions from a cytokine-rich to a cytokine-free environment. Totest this idea, the inventors used a cytokine pulse assay. Tc1 and Tc0cells were cultured overnight with a saturating dose of IL-2 or IL-15,washed, and replated without cytokine as shown in FIG. 2e . Consistentwith the hypothesis, only pre-culture of Tc1 cells with IL-2 led tosustained STAT5 phosphorylation in the absence of additional cytokine(FIGS. 11a-11e ). To directly test the role of IL-2Rα in promotingsustained signaling on effector CD8⁺ T cells, the inventors cultured Tc1cells for 90 minutes with IL-2 in the absence or presence of blockinganti-IL-2Rα antibody (PC61 clone). This shorter pulse was equallysufficient for inducing sustained signaling as indicated by STAT5phosphorylation (FIG. 2f ). Importantly, blockade of IL-2Rα completelyabolished the sustained IL-2 signaling as indicated by STAT5phosphorylation and proliferation (FIGS. 2f , 12, and 13 a-13 b).Polyclonal effector CD8⁺ T cells activated in the absence of IL-12 alsoshowed sustained IL-2 signaling, and importantly, effector cellsgenerated from IL-2Rα^(+/−) mice showed roughly half the sustained IL-2signaling (FIG. 2g ). To ensure that these cells had similar IL-2Rβγsignaling potential, the inventors pulsed wildtype and IL-2Rα^(+/−)effector CD8⁺ T cells with IL-15 and found no differences in theirresponse (FIG. 2h ). Notably, the ability to induce sustained IL-2signaling on mouse effector cells was observed with human and mouse IL-2(FIG. 14). Furthermore, culture of human effector T cells with hIL-2 butnot hIL-15 led IL-2Rα-dependent sustained STAT5 phosphorylation (FIGS.15a-15b ). Finally, to verify that IL-2/mAb complexes (clone 5355) usedin the in vivo experiments were permissive to engagement of IL-2Rα, theinventors repeated the pulse assay with hIL-2 and excess anti-IL-2 mAb.In vitro generated IL-2/mAb complexes induced sustained IL-2 signalingthat was dependent on IL-2Rα (FIG. 16a ). In contrast, IL-2/mAb_(CD122)complexes (clone S4B6), which do not engage IL-2Rα (Boyman et al., 2006;Spangler et al., 2015), failed to induce sustained signaling in vitro(FIG. 16b ).

IL-2Rα Facilitates Sustained IL-2 Signaling Through Creation of anExtracellular Reservoir and Recycling.

To understand how IL-2Rα promotes sustained IL-2 signaling, theinventors hypothesized two non-mutually exclusive possibilities. First,IL-2Rα may bind IL-2 and create a cell-surface cytokine reservoir due tothe high ratio of surface IL-2Rα to IL-2Rβγ, as IL-2/IL-2Rαinternalization can only occur in the presence of both IL-2Rβ and γ(Robb and Greene, 1987; Takeshita et al., 1992). Such a reservoir ofIL-2 bound to IL-2Rα would mediate gradual signaling by continuallyfeeding the rate-limiting, endocytosed IL-2Rβγ. In support of thispossibility, the inventors detected high surface levels of IL-2 oneffector CD8⁺ T cells that gradually waned after extended culture, andthis cell-surface IL-2 was dependent on available IL-2Rα (FIGS. 3a-3b ).Furthermore, antibodies against IL-2 added after the removal of freecytokine from IL-2 pulsed cells were able to dampen sustained signaling(FIG. 3c ). A second possible way in which IL-2Rα might sustainsignaling is by promoting recycling of IL-2 from within the cell to thesurface, thus allowing for repetitive signaling. To test thishypothesis, Tc1 cells were pulsed with IL-2 at 37° C. to allow forcytokine internalization. Cells were then stripped of surface IL-2 usingan acid wash. Upon reculture at 37° C., the inventors were able todetect re-appearance of either mIL-2 or hIL-2 on the cell surface (FIG.3d ). Minimal surface IL-2 was observed when cells were pulsed at 4° C.or on the surface of mixed bystander Tc1 cells (FIG. 3e ). Importantly,the species-specificity of the reagents precluded autocrine productionas the source of cell surface IL-2 after acid wash (FIG. 17). Inadditional support of IL-2Rα-mediated recycling, the inventors observedsustained pSTAT5 signaling after acid washing of cells pulsed with hIL-2at 37° C. but not 4° C. (FIG. 3f ). Because internalization of IL-2Rαβγdoes not occur at 4° C., these data provide further support thatsustained signaling occurs in part through an IL-2Rα bound pool ofinternalized IL-2. It is notable that the inventors could not blocksustained STAT5 signaling in cells pulsed with mIL-2 at 4° C. by acidwashing, possibly reflecting a higher affinity of mIL-2 for mIL-2Rαcompared with that of hIL-2 for mIL-2Rα (Spangler et al., 2015; Liu etal., 1996). Finally, confocal microscopy showed discrete punctatestructures of either mIL-2 or hIL-2 when cells were incubated withcytokine at 37° C. but not 4° C. (FIGS. 18a, 18b , and 19). Thesepunctate structures colocalized with IL-2Rα, Rab5, and EEA1, but lessfrequently with LAMP-1, consistent with intracellular IL-2 beingaccessible to the recycling pathway (FIGS. 3g, 3h and 20a-20c ) (Grantand Donaldson, 2009; Mu et al., 1995). Taken together, these resultssuggest that IL-2Rα both promotes an extracellular reservoir for IL-2and mediates recycling of IL-2.

IL-2Rα Expression on Donor CD8⁺ T Cells Provides a Competitive Advantageto IL-2 Therapy in a Lymphoreplete but not Lymphopenic Host Environment.

The results thus far suggest that the differential responsiveness of Tc1cells to IL-2- and IL-15 therapy in vivo is a consequence of IL-2Rα ondonor T cells providing a competitive advantage to accessing cytokine.To formally test this hypothesis, the inventors initially attempted toactivate T cells from wildtype and IL-2Rα^(−/−) mice. However, thisproved technically not feasible for us as T cells isolated fromIL-2Rα^(−/−) mice were resistant to normal activation, likely due to theimmune alterations in the absence of IL-2 responsiveness (Willerford etal., 1995). Therefore, the inventors used polyclonal IL-2Rα^(+/−) Tcells, as these cells activated comparably to wildtype T cells and hadapproximately half the expression of IL-2Rα (FIG. 4a ). Using the Thy1.1congenic marker to distinguish between genotypes, these two cellpopulations were mixed and adoptively transferred into non-irradiatedB6(CD45.1) recipient mice. Mice were treated with IL-2/mAb orIL-15/sIL-15Rα for 1 week. The inventors hypothesized that IL-2Rα^(+/−)donor CD8⁺ T cells would not persist as well as their wildtypecounterparts due to loss of one allele. In contrast to the expectations,wildtype and IL-2Rα^(+/−) donor T cells did not show differentialresponsiveness to treatment with IL-2/mAb or IL-15/sIL-15Rα complexes(FIG. 4a-4b ). These results suggest a threshold of IL-2Rα in vivo, bothin terms of level and durability of expression, that when reached issufficient for providing donor cells a competitive advantage to IL-2therapy in a lymphoreplete environment.

As an alternative means of assessing the role of IL-2Rα on donor T cellsin vivo, the inventors compared the responsiveness of IL-2Rα^(hi) donorT cells to IL-2- and IL-15 therapy with the addition of lymphodepletionto destroy host cells. The inventors predicted that the advantage ofIL-2Rα-competent cytokine therapy would be lost in the absence of hostIL-2Rβγ⁺ lymphocytes competing for cytokine (FIG. 21a ). Thus, mice weregiven total body irradiation (600 rad) prior to adoptive transfer ofeffector Tc1 CD8⁺ T cells, and then treated for one week with IL-2/mAband IL-15/sIL-15Rα complexes. Consistent with the prediction, both IL-2and IL-15 therapy effectively augmented the persistence of donor cellsboth in the blood and in the spleen, and only in lymphodepleted mice(FIGS. 4c and 21b-c ). These results demonstrate a critical role forIL-2Rα on donor T cells in promoting IL-2 responsiveness in alymphoreplete host environment.

It was also shown that low-dose IL-2 leads to preferential expansion ofadoptively transferred donor tumor-reactive T cells by engagement ofIL-2Rα (FIG. 23). B6 mice were injected with 250,000 B16-F1 tumor cells(s.c.). Eight days later, mice were adoptively transferred with 3×10⁶tumor-reactive activated T cells (pmel-1) conditioned with IL-12 toinduce high levels of IL-2Rα. On the day of adoptive T cell transfer, 2days later, and 4 days later, mice were treated with hIL-2 (1.5 ug),hIL-2/mAb complexes (1.5 ug hIL-2 and 7.5 ug anti-hIL-2 mAb (MAB602)),or hIL-15/sIL-15Rα-Fc complexes (0.5 ug hIL-15+2.3 ug sIL-15Rα-Fc). Onday 6 after adoptive transfer, mice were bled and the frequency of donorT cells (CD8⁺ Thy1.1⁺) in the peripheral blood was determined.

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   Amano et al., “A hydrophobic amino acid cluster inserted into the    C-terminus of a recycling cell surface receptor functions as an    endosomal sorting signal,” BBRC, 441:164-168, 2013.-   Boyman et al., “Selective Stimulation of T Cell Subsets with    Antibody-Cytokine Immune Complexes,” Science, 311:1924-1927, 2006.-   Boyman et al., Selective stimulation of T cell subsets with    antibody-cytokine immune complexes. Science 311, 1924-1927, 2006.-   Grant and Donaldson, Pathways and mechanisms of endocytic recycling.    Nature reviews. Molecular cell biology 10, 597-608, 2009.-   Lisiero et al., Enhanced sensitivity to IL-2 signaling regulates the    clinical responsiveness of IL-12-primed CD8(+) T cells in a melanoma    model. Journal of immunology 186, 5068-5077, 2011.-   Liu et al., The alpha chain of the IL-2 receptor determines the    species specificity of high-affinity IL-2 binding. Cytokine 8,    613-621, 1996.-   Mu et al., EEA1, an early endosome-associated protein. EEA1 is a    conserved alpha-helical peripheral membrane protein flanked by    cysteine “fingers” and contains a calmodulin-binding IQ motif. J    Biol Chem 270, 13503-13511, 1995.-   Robb and Greene, Internalization of interleukin 2 is mediated by the    beta chain of the high-affinity interleukin 2 receptor. The Journal    of experimental medicine 165, 1201-1206, 1987.-   Rubinstein et al., “Converting IL-15 to a superagonist by binding to    soluble IL-15R{alpha},” Proc Natl Acad Sci USA., 103(24):9166-71,    2006.-   Rubinstein et al., Converting IL-15 to a superagonist by binding to    soluble IL-15R{alpha}. Proceedings of the National Academy of    Sciences of the United States of America 103, 9166-9171, 2006.-   Rubinstein et al., Ex vivo interleukin-12-priming during CD8(+) T    cell activation dramatically improves adoptive T cell transfer    antitumor efficacy in a lymphodepleted host. Journal of the American    College of Surgeons 214, 700-707; discussion 707-708, 2012.-   Rubinstein et al., IL-7 and IL-15 differentially regulate CD8+    T-cell subsets during contraction of the immune response. Blood 112,    3704-3712, 2008.-   Shin et al., “Epigenetic Modifications Induced by Blimp-1 Regulate    CD8+ T Cell Memory Progression during Acute Virus Infection,”    Immunity, 39:661-675, 2013.-   Spangler et al., Antibodies to Interleukin-2 Elicit Selective T Cell    Subset Potentiation through Distinct Conformational Mechanisms.    Immunity 42, 815-825, 2015.-   Stoklasek et al., Combined IL-15/IL-15Ralpha immunotherapy maximizes    IL-15 activity in vivo. Journal of immunology 177, 6072-6080, 2006.-   Takeshita et al., Cloning of the gamma chain of the human IL-2    receptor. Science 257, 379-382, 1992.-   Teege et al., Tuning IL-2 signaling by ADP-ribosylation of CD25.    Scientific reports 5, 8959, 2015.-   Willerford, et al. Interleukin-2 receptor alpha chain regulates the    size and content of the peripheral lymphoid compartment. Immunity 3,    521-530, 1995.

1. An isolated transgenic cell comprising: (i) an elevated surfaceexpression level of at least one cytokine receptor or cytokine receptorco-stimulator, relative to an activated T-cell; or (ii) a nucleic acidmolecule encoding at least one cytokine receptor or cytokine receptorco-stimulator polypeptide comprising a mutation that increases theactivity of the receptor.
 2. The isolated transgenic cell of claim 1,wherein the at least one cytokine receptor is the IL-2 or IL-15receptor.
 3. The isolated cell of claim 1, wherein the at least onecytokine receptor is IL-12Rβ1, IL-12Rβ2, IL-6Rα, GP130, IL-4Rα, IL-2Rγ,IL-21Rα, IL-2Rγ, IL-27R, GP130, IL-2Rβ2, GP130, IL-12Rβ1, IL-23R, IL-9Rαand/or IL-2Rγ. 4-10. (canceled)
 11. The isolated cell of claim 1,wherein the at least one cytokine receptor co-stimulator is ICOS, 4-1BB,CD28, G-CSFR, GM-CSFRα, or GM-CSFRβc. 12-15. (canceled)
 16. The isolatedcell of claim 1, wherein the cell is a human cell.
 17. The isolated cellof claim 1, wherein the cell is a T-cell.
 18. The isolated cell of claim17, wherein the T-cell is a T-cell targeted to a cancer cell antigen.19-23. (canceled)
 24. The isolated cell of claim 18, wherein the cancercell antigen is GP240, 5T4, HER1, CD-33, CD-38, VEGFR-1, VEGFR-2, CEA,FGFR3, IGFBP2, IGF-1R, BAFF-R, TACI, APRIL, Fn14, EGFR, ERBB2, ERBB3CD19, CD20 or mesothelin.
 25. The isolated cell of claim 1, wherein thecell expresses a chimeric antigen receptor (CAR) or a recombinant T-cellreceptor (TCR). 26-28. (canceled)
 29. The isolated cell of claim 2,wherein the IL-2 receptor is IL-2Rα.
 30. The isolated cell of claim 2,wherein the IL-2 receptor is IL-2Rβ or IL-2Rγ.
 31. The isolated cell ofclaim 2, wherein the mutation increases surface expression, increasesstability or increases ligand binding of the IL-2 receptor polypeptide.32-37. (canceled)
 38. The isolated cell of claim 1, comprising thenucleic acid molecule encoding cytokine receptor polypeptide operablylinked to a heterologous promoter. 39-40. (canceled)
 41. The isolatedcell of claim 39, wherein the heterologous promoter is a ligandinducible promoter. 42-61. (canceled)
 62. A method of producingtherapeutic cells comprising: (i) selecting a population of cells havingan increased activity of at least a one cytokine receptor; and (ii)culturing the cells in the presence of a ligand for said at one cytokinereceptor.
 63. The method of claim 62, wherein the at least one cytokinereceptor is a IL-2R. 64-72. (canceled)
 73. A method of treating adisease comprising: transferring at least one receptor gene into atleast one cell, and treating said cell or cells with an agonist of thereceptor transcribed by said receptor gene.
 74. (canceled)
 75. Themethod of claim 73, wherein the receptor gene is Interleukin-2 receptoralpha (IL-2Rα).
 76. The method of claim 73, wherein the agonist isInterleukin-2 (IL-2).
 77. The method of claim 73, wherein the disease iscancer. 78-98. (canceled)